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Fungal Genetics and Biology 70 (2014) 42–67
Contents lists available at ScienceDirect
Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi
Review
Fungal model systems and the elucidation of pathogenicity determinants
Elena Perez-Nadales a,⇑, Maria Filomena Almeida Nogueira b, Clara Baldin c,d, Sónia Castanheira e, Mennat El Ghalid a, Elisabeth Grund f, Klaus Lengeler g, Elisabetta Marchegiani h, Pankaj Vinod Mehrotra b, Marino Moretti i, Vikram Naik i, Miriam Oses-Ruiz j, Therese Oskarsson g, Katja Schäfer a, Lisa Wasserstrom g, Axel A. Brakhage c,d, Neil A.R. Gow b, Regine Kahmann i, Marc-Henri Lebrun h, José Perez-Martin e, Antonio Di Pietro a, Nicholas J. Talbot j, Valerie Toquin k, Andrea Walther g, Jürgen Wendland g
a Department of Genetics, Edificio Gregor Mendel, Planta 1. Campus de Rabanales, University of Cordoba, 14071 Cordoba, Spain b Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK c Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI), Beutembergstr. 11a, 07745 Jena, Germany d Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Beutenbergstr. 11a, 07745 Jena, Germany e Instituto de Biología Funcional y Genómica CSIC, Universidad de Salamanca, 37007 Salamanca, Spain f Functional Genomics of Plant Pathogenic Fungi, UMR 5240 CNRS-UCB-INSA-Bayer SAS, Bayer CropScience, 69263 Lyon, France g Carlsberg Laboratory, Department of Yeast Genetics, Gamle Carlsberg Vej 10, DK-1799, Copenhagen V, Denmark h Evolution and Genomics of Plant Pathogen Interactions, UR 1290 INRA, BIOGER-CPP, Campus AgroParisTech, 78850 Thiverval-Grignon, France i Max-Planck-Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany j School of Biosciences, Geoffrey Pope Building, University of Exeter, Exeter EX4 4QD, UK k Biochemistry Department, Bayer SAS, Bayer CropScience, CRLD, 69263 Lyon, France

article info
Article history: Received 3 February 2014 Accepted 25 June 2014 Available online 7 July 2014
Keywords: Fungal model organism Plant fungal pathogen Human fungal pathogen Genomics Virulence

abstract
Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity. Ó 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2. Aspergillus fumigatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.1. Overview of the A. fumigatus genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.2. Assessing virulence of A. fumigatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.3. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.3.1. Virulence determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.3.2. Signaling involved in virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.3.3. Host perception and response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.3.4. Development of anti-infective strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 2.3.5. Contribution of fungal secondary metabolism to virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
⇑ Corresponding author. Fax: +34 957212072.
E-mail address: [email protected] (E. Perez-Nadales).
http://dx.doi.org/10.1016/j.fgb.2014.06.011 1087-1845/Ó 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

E. Perez-Nadales et al. / Fungal Genetics and Biology 70 (2014) 42–67

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2.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 3. Candida species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.1. Comparative genomics in Candida species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.2. The toolbox for C. albicans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.3. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.3.1. Genome functional analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.2. Altered host responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.3. Yeast-to-hypha transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.3.4. Importance of the cell wall in immune recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 4. Fusarium oxysporum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 4.1. Overview of the F. oxysporum genome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.2. Assessing virulence of F. oxysporum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.3. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.3.1. Role of MAPK cascades in virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.3.2. Lineage specific (LS) chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4.3.3. Secreted effectors and gene-for-gene system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4.3.4. F. oxysporum as a model for fungal trans-kingdom pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5. Ashbya gossypii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5.1. Comparative genomics in Eremothecium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5.2. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5.2.1. Riboflavin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5.2.2. Hyphal growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5.2.3. Septation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5.2.4. Nuclear division and movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5.2.5. Genome evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5.3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 6. Magnaporthe oryzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 6.1. Overview of the M. oryzae genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 6.2. Assessing virulence of M. oryzae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 6.3. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.3.1. Mechanisms of fungal infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.3.2. Plant pathogen interactions in the rice blast fungus M. oryzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.3.3. Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 7. Ustilago maydis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 7.1. Comparative genomics in U. maydis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 7.2. Assessing virulence of U. maydis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 7.3. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 7.3.1. Host perception and plant response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 7.3.2. Effector function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 7.3.3. Cell cycle, dimorphism and pathogenic development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 7.3.4. Signaling and MAP kinase targets involved in virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 7.3.5. Understanding the molecular basis of fundamental eukaryotic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 7.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 8. Zymoseptoria tritici . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 8.1. Overview of the Z. tritici genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 8.2. Assessing virulence of Z. tritici . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 8.3. Current research interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 8.3.1. Signaling pathways and protein secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 8.3.2. Plant-pathogen interaction: effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 8.3.3. Disease control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 8.3.4. Evolutionary genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 8.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 9. General conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Appendix A. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

1. Introduction
A small fraction of the estimated 5 million fungal species are responsible for devastating diseases affecting agriculture and human health. Emerging infectious diseases (EIDs) caused by fungi are increasingly recognized as major threats to food security and animal health (Brown et al., 2012; Fischer et al., 2008). Dispersal and emergence of fungal diseases are furthermore promoted by

human activity, primarily through global trade which lacks sufficient biosecurity measures, and may be exacerbated by the impact of climate change (Brasier, 2008; Fisher et al., 2012; Gange et al., 2007; Harvell et al., 1999; Ratnieks and Carreck, 2010; Verweij et al., 2009). Fungal pathogens are characterized by a remarkable genetic flexibility that facilitates rapid evolution and adaptation to the host or environment (Calo et al., 2013). These features, when combined with Darwinian selection, support the emergence of new

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lineages with increased virulence or altered host range (Croll and McDonald, 2012). This highly adaptive behavior is promoted by an immense fungal genome plasticity (Calo et al., 2013). Besides sexual reproduction, parasexualilty (Pontecorvo, 1956), aneuploidy (Selmecki et al., 2006), transposons, telomere instability (Starnes et al., 2012) or horizontal gene transfer (Ma et al., 2010; Richards et al., 2011) amongst others enable adaptive evolution by promoting mitotic recombination, independent chromosomal assortment, gain or loss of chromosomes and translocations. Moreover, fungi exhibit morphogenetic plasticity which enables them to colonize and invade tissue as hyphae, which primarily extend at their tips (Fischer et al., 2008), while often forming differentiated spores, infection structures, fruiting bodies and unicellular, yeast cells, that can aid rapid dispersal (Sudbery et al., 2004).
In this review, we cover an array of fungal species, encompassing diverse taxonomic groups, that cover several hundred million years of evolution (Fig. 1). We include two major fungal pathogens of humans, Aspergillus fumigatus and Candida albicans, a facultative pathogen of both animals and plants, Fusarium oxysporum, and plant pathogenic species Magnaporthe oryzae, Mycospherella graminicola, Ashbya gossypii and Ustilago maydis which display infection-associated dimorphism and cellular differentiation. Collectively, this provides an overview of diverse mechanisms of pathogenesis and some unifying themes, while also encompassing some of the best-studied and understood fungal pathosystems.
The purpose of the review is to highlight both specific differences and unifying features of some of the best-studied fungal pathogens and to highlight the significant challenges that remain in developing a deep understanding of fungal pathogenesis.
2. Aspergillus fumigatus
Aspergillus fumigatus is a filamentous fungus that can be isolated from compost soil, where it proliferates in organic debris and plays

an essential role in carbon and nitrogen recycling. Fumigatus is the most important air-borne fungal pathogen and can grow at temperatures up to 55 °C, while its spores survive temperatures of up to 70 °C (Brakhage and Langfelder, 2002). The fungus propagates asexually with release of thousands of spores per conidial
head into the atmosphere. Due to their small size (2–3 lm diame-
ter), the spores can easily reach lung alveoli (Latge, 2001). A first description of pulmonary aspergillosis was published in
1842 by physician John H. Bennett (Supplementary Fig. 1A), who noted the presence of a fungus in the lungs of a post mortem patient with pneumothorax. Almost 50 years elapsed, however, before A. fumigatus was recognized as the primary cause of the infection (Barnes, 2004). Today, A. fumigatus conidia infect millions of susceptible individuals, causing allergies associated with asthma, allergic sinusitis and bronchoalveolitis (Denning et al., 2013). In cavities in the lungs of tuberculosis patients, A. fumigatus spores germinate and develop into a fungus ball, or non-invasive aspergilloma (Riscili and Wood, 2009). In addition to these forms of aspergillosis, which are not life-threatening, patients with altered immune status such as leukemia patients or transplant patients are at risk to develop invasive aspergillosis (IA), with an estimated number of more than 200,000 cases per year (Brown et al., 2012; Garcia-Vidal et al., 2008). Mortality rates for IA reach around 50% of the cases when patients are treated and increase to more than 90% when the diagnosis is missed or delayed (Brown et al., 2012).
2.1. Overview of the A. fumigatus genome
A first draft of the A. fumigatus clinical isolate Af293 genome was published in 2005 (Nierman et al., 2005). Eight chromosomes were identified containing about 10,000 genes (Table 2). Three years later, the genome sequence of a second A. fumigatus isolate, A1163, was released (Fedorova et al., 2008). Comparative analysis showed conservation of 98% of the sequence between the two

Fig. 1. Phylogenetic tree. Fungal species phylogeny generated from a concatenated alignment of 49 conserved fungal genes using maximum likelihood. The tree covers 21 taxa and 9033 amino acid positions. Bootstrap values for each node are reported as percentages. To generate the tree, protein sequences of the 49 selected conserved single copy genes from the 21 fungal species were aligned using Clustal W (Larkin et al., 2007) and the obtained conserved sequence blocks were sampled with G-blocks (Talavera and Castresana, 2007). All the aligned sequences were then concatenated to one file using Galaxy (Goecks et al., 2010). Finally, PhyML (Guindon et al., 2010) was used to generate the phylogenetic tree with 100 bootstraps for branch support and LG as the amino acid substitution model as identified by ModelGenerator (Keane et al., 2006) (Keane et al., 2006). The tree was visualized using TreeDyn (Chevenet et al., 2006).

Table 1 Overview of biological features.

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Species

Human fungal pathogens

Plant fungal pathogens

Aspergillus fumigatus Candida albicans

Magnaporthe oryzae

Fusarium oxysporum

Zymoseptoria tritici

Ustilago maydis

Ashbya gossypii

Taxonomy

Phylum: Ascomycota Class: Eurotiomycetes
Order: Eurotiales Family: Trichocomaceae Genus: Aspergillus
Species: A. fumigatus

Phylum: Ascomycota Subphylum: Saccharomycotina Class: Saccharomycetes Order: Saccharomycetales
Family: Saccharomycetaceae Genus: Candida Species: C. albicans

Phylum: Ascomycota Class: Sordariomycetes
Order: Magnaporthales Family: Magnaporthaceae
Genus: Magnaporthe
Species: M. oryzae

Phylum: Ascomycota Class: Sordariomyctes
Order: Hypocreales Family: Nectriaceae
Genus: Fusarium
Species: F. oxysporum

Phylum: Ascomycota Class: Dothideomycetes
Order: Capnodiales Family: Mycosphaerellaceae Genus: Zymoseptoria
Species: Z. tritici

Phylum: Basidiomycota Class: Ustilaginomycetes
Order: Ustilaginales Family: Ustilaginaceae
Genus: Ustilago
Species: U. maydis

Phylum: Ascomycota Subphylum: Saccharomycotina Class: Saccharomycetes Order: Saccharomycetales
Family: Saccharomycetaceae Genus: Eremothecium Species: A. gossypii

Predominant cell-type Multinucleate; septated filaments

Budding yeast, pseudohyphae and true hypha (in which elongated yeast cells remain attached after cytokinesis)

Filamentous mycelium

Filamentous mycelium, Microconidia

Dimorphic fungus: yeastlike cells/filamentous mycelium

Unicellular budding yeast

multinucleate; septated filaments

Sexual cycle

Yes, but in nature predominantly asexual

Parasexual cycle (mating of diploid cells followed by mitosis and chromosome loss instead of meiosis

Yes, but in nature predominantly asexual

Not identified

Yes, occurs during epidemics

Yes, occurs only inside the not identified plant

Mating-type system

Bipolar heterothallism MATa and MATa
MAT1-1, MAT1-2

Bipolar heterothallism MAT1-1, MAT1-2

MAT1 gene identified and Bipolar heterothallism expressed in F. oxysporum. A MAT1-1, MAT1-2 mixed distribution of MAT1-1 and MAT1-2 alleles in Fusarium species complex

Tetrapolar: a locus (2

bipolar; MATa/a

alleles) b locus ( 20 alleles)

Spores

Uninucleate conidia and binucleate ascospores

Chlamydospore

Conidia (asexual spore) and Microconidia, ascospores (sexual spore) macroconidia,
clamydospores

Ascospores and pycnidiospores

Diploid spores, teliospores Ascospores

Pathogenicity

Animals (can cause asthma, aspergilloma, invasive aspergillosis)

Candidiasis (skin infections) Rice and some and Candidemia (presence monocotylous plants (e. g. of Candida albicans in blood) barley, wheat) in immuno-compromised individuals

Fusarium wilt on plant crops Emerging cause of fusariosis in humans

Bread and durum wheat Corn and teosinte plants (septoria tritici leaf blotch)

Cotton/citrus fruits

Other features

Toxin production

Homothallic and heterothallic mating and haploid mating also possible, but rare

Fusarium species complex are pathogenic especially to agriculture plants

Highly resistant to UV radiation

Riboflavin overproducer

45

46

Table 2 Overview of fungal genome data.

E. Perez-Nadales et al. / Fungal Genetics and Biology 70 (2014) 42–67

Species Genome size (Mb) Chromosomes GC content (%) Number of Genes Non-coding RNAs (tRNAs) Introns Avg. gene size/intergenic
region Transposons
S. cerevisiae homology Mitochondrial DNA (kb) References

Human fungal pathogens
Aspergillus fumigatus
29.420 8 50.0 9783 179 Average 1.8 per gene

Candida albicans
14.88 8 33.3 6354 132 415 introns in the entire genome

1.64 kb/1.22 kb

1.47 kb/858 bp

Plant fungal pathogens Magnaporthe oryzae 41.03 7 51.6 12827 325 Average 1.8 per gene
2 kb/1.4 kb

Fusarium oxysporum 61.36 15 48.4 17708 308 Unknown
1.3 kb

Zymoseptoria tritici Ustilago maydis

39.7 21 51.7 10900 Unknown Average 1.5 per gene
P1.15 kb

20.50 23 57.0 6788 104 3093 introns in the entire genome, average 0.46 per gene 1.74 kb/973 bp

Ashbya gossypii
8.76 7 51.8 4726 192–293 (216) 226 introns in the entire genome
1.9 kb/340 bp

8 (Predicted DDE1 transposon-related ORF & putative transposase, induced by exposure to human airway epithelial cells) Predicted LINE (long interspersed nuclear elements), LINE-like reverse transcriptase
Predicted gypsy transposon- related ORF Predicted mariner Ant1 transposon-related ORF 60% 32 Nierman et al. (2005)
Fedorova et al. (2008)

3 (Zorro3-R ZORRO1, Zorro2-1; member of L1 clade of transposons and encodes a potential DNAbinding zinc-finger protein)
64% 41 Braun et al. (2005) Mitrovich et al. (2007)

9.7% of genome comprises repeated sequences longer than 200 base pairs (bp) and with greater than 65% similarity
36.9% 34.95 http://www.broadinstitute.org Dean et al. (2005)

28% of the genome identified as repetitive sequence
Retroelements (copia-like and gypsy-like, LINEs (long interspersed nuclear elements) and SINEs (short interspersednuclear elements). DNA transposons (Tc1-mariner, hAT-like, Mutator-like, and MITEs)
Unknown 34.48 http:// www.broadinstitute.org Rep and Kistler (2010)

20%
22.5% 44 Orton et al. (2011) Goodwin et al. (2011)

1.1% DNA with similarity to transposons
(hobS, tigR retroelements)
33% (20% identity cut-off) 56.8 Kamper et al., 2006

no -
95% 23.5 Dietrich et al. (2004)

E. Perez-Nadales et al. / Fungal Genetics and Biology 70 (2014) 42–67

47

isolates, with the majority of the remaining 2% being strain-specific genes clustered in blocks from 10 to 400 kb in size, including a large number of pseudogenes and repeat elements (Fedorova et al., 2008). Comparison of the sequenced A. fumigatus genomes with those of closely related species such as Neosartorya fischeri and Aspergillus clavatus showed the presence of lineage-specific genes often localized to the sub-telomeric regions. These genomic islands were suggested to represent a gene repository that could play a role in adaptation to different environments, such as compost or a living host (Fedorova et al., 2008). Transcriptome studies based on RNA-Seq and proteome studies are routinely carried out with A. fumigatus (Horn et al., 2012; Kroll et al., 2014). The updated features of the A. fumigatus genome are available at the Aspergillus Genome Database website (Table 3).
2.2. Assessing virulence of A. fumigatus
To understand the infection process of A. fumigatus and identify fungal factors involved in pathogenesis, appropriate infection models are required. Mouse infection models have been crucial for the study of pathogenicity, for the characterization of hostpathogen interactions and for development of therapeutic approaches (Clemons and Stevens, 2005). According to the immunosuppression regimen, mouse infection models are divided into neutropenic or non-neutropenic models (Liebmann et al., 2004a). Because it is believed that infection starts by inhalation of conidia, in mouse infection models, conidia are administered by intranasal, intra-tracheal inoculation or inhalation. Mouse models however, have disadvantages, including an open question about their reliability as models for human diseases (Seok et al., 2013), as well as increasing public concern regarding animal welfare and the clinical value of animal research, which is resulting in increasing regulatory demands. This, together with the requirement for costly facilities and personnel, is driving the development of alternative infection models for fungal pathogens. The larvae of the greater wax moth Galleria mellonella, for example, provide important advantages. They are readily available from suppliers at convenient cost, they are large enough (around 2.5 cm in length) to be easily inoculated (Mylonakis, 2008) and can be maintained at 37 °C, which means that infections can be studied at the temperature at which they occur in humans (Mylonakis, 2008). However, results obtained with such models are of limited value because there are significant differences in the immune responses of invertebrates and mammals. Embryonated chicken eggs have also been used as an alternative infection model for analysis of fungal pathogenicity in A. fumigatus, with promising results (Jacobsen et al., 2010).
2.3. Current research interests
2.3.1. Virulence determinants It is debated whether A. fumigatus has true virulence factors or
simply physiological characteristics, such as thermo-tolerance, that enable the fungus to grow in an immuno-compromised human host. Several virulence determinants of A. fumigatus have, however, been characterized. These determinants include the siderophore-mediated iron uptake system (Schrettl et al., 2004), and the pksP gene, which is involved in the biosynthesis of dihydroxynaphthalene (DHN) melanin, which forms the gray-green spore pigment (Heinekamp et al., 2012; Horn et al., 2012; Langfelder et al., 1998; Thywissen et al., 2011; Volling et al., 2011). DHN melanin inhibits both apoptosis and acidification of conidia-containing phagolysosomes of macrophages (Thywissen et al., 2011; Volling et al., 2011). Mechanistically, it was reported that DHN melanin inhibited assembly of v-ATPase on the phagosomal membrane (Heinekamp et al., 2012). A. fumigatus also possesses immune-evasion mechanisms which reduce recognition of both immune

effector cells and the complement system (Aimanianda et al., 2009; Behnsen et al., 2008, 2010; Horn et al., 2012). It can be expected that virulence is a multifactorial process and thus more virulence-associated traits will be discovered.
2.3.2. Signaling involved in virulence Several genes of the cAMP signal transduction pathway are
required for pathogenicity. These include genes encoding adenylate cyclase (AcyA), protein kinase A (PKA) and a stimulating Ga protein-encoding gene designated gprA (Liebmann et al., 2004b; Zhao et al., 2006). Recently, the cAMP signaling pathway has also been related to regulation of the DHN melanin production (Abad et al., 2010; Grosse et al., 2008).
The calcineurin/calmodulin signaling pathway is required for formation of cell shape and pathogenicity in A. fumigatus. Deletion of the gene encoding the catalytic subunit of calcineurin phosphatase (calA) resulted in a branching defect and limited growth, as well as attenuation of virulence (da Silva Ferreira et al., 2007; Juvvadi et al., 2013). A target of calcineurin is the zinc finger transcription factor CrzA, which is implicated in germination, polarization, cell wall structure, asexual development and virulence (Cramer et al., 2008). Of the four MAPKs found in A. fumigatus, it was shown that MpkA regulates the cell wall integrity pathway and SakA the hyperosmotic glycerol pathway (Jain et al., 2011; May et al., 2005; Rispail et al., 2009).
Two GPCRs were shown to be required for virulence in A. fumigatus: GprC and GprD (Gehrke et al., 2010). Little is known, however, about the downstream multi-subunit G-proteins. A.
fumigatus encodes only three Ga subunits, while other Aspergilli harbor up to four Ga proteins. For gpaB, most likely involved in
cAMP signal transduction, its involvement in pathogenicity was demonstrated (Liebmann et al., 2004b; Rispail et al., 2009).
The seven transmembrane domain (7TMD) protein PalH was recognized as a putative pH sensor and also shown to be required for virulence on mice (Grice et al., 2013). Three histidine kinase receptors TcsA/Fos-1, TcsB and TcsC have been characterized. Deletion of tcsA led to a strain with reduced virulence in a systemic murine model while the only phenotype detectable for DtcsB was reduced sensitivity to SDS (Grice et al., 2013). TcsC has been shown to play a role in the high osmotic pressure response, and growth in the presence of nitrate as nitrogen source (McCormick et al., 2012). Finally, a number of a cell wall stress sensors have been described, including Wsc1, Wsc2, Wsc3 and MidA. Wsc1 is involved in resistance against echinocandins. MidA appears to be essential for thermotolerance at elevated temperatures and to counteract the effects of cell wall-disrupting compounds such as congo red and calcofluor white. Wsc1, Wsc3 and MidA show also some overlapping roles in radial growth and conidiation. The role of Wsc2 remains to be elucidated (Dichtl et al., 2012). Another conserved signal cascade is the CPC system, which links environmental stresses to amino acid homeostasis. Deletion of the transcriptional activator CpcA led to reduced virulence, while deletion of the sensor kinase CpcC resulted in increased sensitivity towards amino acid starvation (Abad et al., 2010).
2.3.3. Host perception and response In healthy individuals, inhaled conidia of A. fumigatus are rap-
idly attacked by the immune system of the host. Physical barriers of the lung and the innate immunity are of major importance for defense. Epithelial cells of the upper respiratory tract contribute to elimination of microbes through secretion of mucus and ciliamediated active transport (Brakhage, 2005). In alveoli, there are epithelial cells, called pneumocytes, responsible for secretion of a pulmonary surfactant with antimicrobial activity (Balloy and Chignard, 2009). A decisive role for elimination of pathogens is played by macrophages and neutrophils. Alveolar macrophages

Table 3 Overview of molecular tools.

Species Transformation

Human fungal pathogens Aspergillus fumigatus Protoplasts & electroporation

Minimal homology for gene deletion
Episomal elements

>500 bp no

Promoters [c]

[r] Pyomelanin promoter, PTet

constitutive [r] system, Xylose-regulated

regulatable

promoter

Commonly used selection markers

Hygromycin B, Pyrithiamine, Phleomycin

Reporter genes lacZ, GFP

Fluorescent

GFP, mCherry

protein labels

Cytochemical dyes DAPI, Calcofluor White, Mito

tracker

Arrays

Affimetrix, Febit,

Plant fungal pathogens

Candida albicans

Magnaporthe oryzae

Lithium acetate, spheroplast PEG/CaCl2 & ATMT

fusion & electroporation (Agrobacterium-mediated

transformation)

50 bp

500 bp

Fusarium oxysporum Protoplasts & ATMT
>1000 bp

Zymoseptoria tritici ATMT
500 bp

No episomal plasmids but integrative plasmids include CIp10 (artificial integration at RPS10 locus), pDUP/PDIS (shuttle vector for integration at NEUT5 l) [c] PADH1, [r] PACT1, [r]
PTEF3, [c] PEF1-a2, [r]
PGAL1, [r] PPCK1, [r] PSAP2, [r] PMRP1, [r] PHEX1, [r] PMET3, [r] PMAL2, [r] PTET, [r] PSAT1 URA3, LEU2, HIS1, ARG4, MH3, Nourseothricin, FLP and Cre-Lox technology
ADH1, ACT1, TEF3, EF1-a2,
GAL1, PCK1, SAP2, MRP1, HEX1, MET3, MAL2, TET, SAT1 mCherry, GFP, YFP, CFP, RFP, Venus Calcoflour White, Alcian Blue Custom made DNA microarray, Affymetrix

no
[r] PICL1 [r] PNiA1 [c] PMPG1
Hygromycin B, Sulfonylurea, Glufosinate, Geneticin G418, Nourseothricin GFP, GUS, HcRed
GFP, RFP, mCherry, YFP Calcofluor White, DAPI, FM4-64, WGA Custom made DNA microarray, Affymetrix

self-replicative ARS (active replicating system) plasmid pFNitLam-Tlam, linear
[r] Thiamine repressed Psti35 promoter [c] PgpdA
Hygromycin B, Phleomycin
GFP, ChFP
GFP; ChFP Calcofluor White, DAPI, FITC Custom made DNA microarray, Affymetrix

no
[c] Pacu-3 [c] Pgpd
Hygromycin B, Bialaphos, Geneticin G418, Carboxin GFP
GFP DAPI, Calcofluor White, WGA alexa, Mito tracker Custom made DNA microarray, Affymetrix

Pathogenicity models

Roche, TIGR (RNA-seq mostly replacing the array platforms) Animal models: mouse and embryonated chicken eggs

Reconstituted epithelial models: chick chorioallantoic model

Rice, barley

Non-mammalian models: C. elegans, D. melanogaster, G. mellonella, B. mori, Zebrafish Mammalian models: murine intravenous model, murine gastrointestinal colonization and dissemination model

Plant models:Tomato plants, tomato fruits, apple fruits; Mammalian model: immunodepressed mice;. Invertebrate model: G. mellonella

Wheat

Ustilago maydis Protoplasts
500 bp
UmARS plasmids

Ashbya gossypii Electroporation & protoplasts 45 bp
ScARS plasmids

[r] PTet system [r] Pnar1, [r] PTEF1, PHIS3; [r] PMET3

Pcrg1; [c] Potef

[r]PTHI13

Hygromycin B, Phleomycin, Carboxin, Nourseothricin, Geneticin G418, FLP technology GFP, GUS

Hygromycin B, AgLEU2, GEN3/KanMX, Nourseothricin
lacZ, GFP

GFP, RFP, mCherry, CFP, YFP Codon optimized

DAPI, FM4-64, Filipin
Custom made DNA microarray, Affymetrix representing 6297 genes

DAPI, FM4-64, Calcofluor White, Mito tracker, Filipin Does not apply

Corn plants

Does not apply

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http://agd.vital-it.ch/ index.html

ATCC10895 CBS102347 and derivatives Ashbya Genome Database

http://genome.jgi.doe.gov/ http://mips.helmholtzMycgr3/Mycgr3.home.html muenchen.de/genre/proj/
ustilago/ http:// www.broadinstitute.org/ annotation/genome/ ustilago_maydis.2/ Home.html

FB1 (a1b1), FB2 (a2b2), FB6a (a2b1), FB6b (a1b2), SG200 (a1mfa2 bE1/bW2), FBD11 (a1a2 b1b2), AB31 (a2 Pcrg1:bE1 Pcrg1:bW2) AB33 (a2 Pnar1:bE1 Pnar1:bW2) Ustilago maydis Database

act very fast and within 30 h can kill 90% of inhaled conidia via phagocytosis (Balloy and Chignard, 2009). Neutrophils form the highest number of intravascular phagocytes. During infection, neutrophils are recruited to alveoli and phagocytose conidia and germlings. The direct killing mechanisms are, however, still unclear. Neutrophils also produce neutrophil extracellular traps (NETs) in response to A. fumigatus, which consist of chromatin covered with granular proteins that display some antimicrobial activity (Bruns et al., 2010).
Host protein receptors are involved in triggering the immune response against A. fumigatus, including secreted complement factors or those exposed on the surface of phagocytes, such as dectin-1 (Steele et al., 2005). As long as inhaled A. fumigatus conidia are covered with a proteinaceous layer formed by hydrophobins, the fungus is masked and immunologically inert (Aimanianda et al., 2009). Once conidia start to germinate, the rodlet layer is not present on the cell surface, leading to exposure of surface components, recognized by immune cells. C-type lectin and Tolllike receptors on host immune cells have, for instance, been proposed to bind to fungal ligands and are involved in activation of the immune response through production of cytokines and chemokines, prostaglandins and reactive oxygen intermediates (Brakhage et al., 2010).

(anamorph: Septoria tritici)

Fusarium Comparative Mycosphaerella graminicola

IPO323

http://www. broadinstitute.org/ annotation/genome/ fusarium_group/ MultiHome.html

F. oxysporum f. sp. lycopersici wild type 4287 (FGSC 9935)

Database

2.3.4. Development of anti-infective strategies The current guideline-recommended therapy against invasive
aspergillosis is the triazole antibiotic voriconazole, which is superior to the toxic polyene amphotericin B deoxycholate (Herbrecht et al., 2002; Steinbach, 2013). While a 2002 clinical trial found an improved response with voriconazole (52.8%) versus amphotericin B (31.6%), the field is still in need of improved antifungals, with entirely novel targets. As recently reported (Steinbach, 2013), a combination therapy of antifungals may be promising, akin to other medical disciplines whereby agents with different mechanisms are employed to provide a synergistic response. In addition, the future holds promise for novel developments such as the generation of immunotherapies based on T cells or dendritic cells. These therapies are based on in vitro proliferation of these cell types upon stimulation by fungus-specific antigens, leading to cell-based vaccination (Lehrnbecher et al., 2013).

http://www. broadinstitute.org/ annotation/genome/ magnaporthe_comparative/ MultiHome.html

Guy11, P1-2, 70-15, PH14, TH3, FR13, BR88 Magnaporthe comparative Database

http://www. candidagenome.org/

SC5314, NGY152, CAI4, RM1000, WP17, SN87, SN95, SN152 Candida Genome Database

2.3.5. Contribution of fungal secondary metabolism to virulence Based on the genome sequence of A. fumigatus, it is estimated
that the fungus encodes at least 39 secondary metabolism gene clusters, which lead to biosynthesis of various low molecular weight compounds. These compounds are often synthesized by non-ribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) (Brakhage, 2013; Inglis et al., 2013). Some of the compounds produced by these secondary metabolism gene clusters have been identified, such as siderophores, DHN melanin and gliotoxin and their involvement in virulence shown (Scharf et al., 2014; Schrettl et al., 2004). It is likely, however, that other compounds of A. fumigatus will contribute to virulence.

CEA10 CEA17 akuB Aspergillus Genome Database http://www.aspgd.org/

2.4. Conclusions

Strains Bioinformatic/
genome databases

In the last 20 years, considerable progress has been made in development of advanced genetic tools for A. fumigatus. While more than 400 mutant strains have been generated (Horn et al., 2012), only a few virulence determinants have been characterized. At present, systems biology approaches are being explored as an potential means to describe the interaction between human host and pathogen. However, the huge amount of data generated by RNA-seq and proteome analyses of both pathogen and host cells, requires development of sophisticated bioinformatic tools. Complete genome-wise gene knock-out libraries would facilitate

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screening efforts such as analysis of the mode of action of antifungal compounds, but are not yet available for A. fumigatus. In parallel, huge efforts are being made to gain insight into virulence mechanisms by analyzing the interaction of A. fumigatus with different cellular systems such as neutrophils, macrophages, epithelial cells and dendritic cells with increasing complexity, such as combining different cellular systems.
To bridge basic research on the pathobiology of A. fumigatus with clinical requirements, a so-called Translational Systems Biology approach has been started which aims to integrate different ‘‘omics’’ data levels and image-based data of host-pathogen interactions into network and spatio-temporal computational models. The main goals are to identify and validate new drugs and new drug targets, increase the efficiency of pathogen identification during an infection and identify effective therapies (Horn et al., 2012).

state (Supplementary Fig. 2B). Homothallic (same sex) mating of diploids has also been reported in strains lacking the Bar1 protease
which inactivates a-pheromone, enabling autocrine signaling
(Alby et al., 2009). Recently, a screen for aneuploid isolates discovered strains that
were fully haploid for all eight chromosomes but retained either a
MLTa or a MLTa locus on chromosome 5. These strains were often
unstable and showed reduced overall fitness but could either mate or autodiploidise to regenerate diploid variants (Hickman et al., 2013). The haploid strains of C. albicans have a full morphogenetic repertoire and have significant potential in development of new genetic strategies including forward genetic screens for analysis of C. albicans biology.
3.1. Comparative genomics in Candida species

3. Candida species
Candida albicans is normally a commensal of the human microflora, but is also a classic opportunistic pathogen causing mucosal thrush, blood stream and systemic infections, termed candidaemia and invasive candidiasis, respectively. Candida infections are normally associated with individuals who are immunocompromised or traumatized due to major surgery, transplantation and invasive medical treatments that disrupt the normal protective bacterial microflora. Five species account for around 95% of all Candida infections, namely C. albicans, C. tropicalis, C. glabrata, C. krusei and C. parapsilosis (Pfaller et al., 2009). Infections are associated with high mortality rates which can vary between 20% and 60%, depending on the specific risk factors and patient group (Calderone and Clancy, 2012).
C. albicans belongs to the Saccharomycetaceae, which includes yeasts, mycelial and pleiomorphic organisms (Table 1). Many of this family can undergo complete sexual cycles but C. albicans has not been demonstrated to undergo meiosis leading to ascospore formation, although most of the S. cerevisiae homologs for meiosis-associated genes are present in its genome.
An important feature of biology of C. albicans is its ability to exist in different morphological forms (Supplementary Fig. 2B) under different environmental conditions. The major vegetative forms of C. albicans are: (i) budding yeast cells, (ii) elongated cojoined yeast cells that undergo synchronous cell division called pseudohyphae and (iii) un-constricted, branching, true hyphae that grow exclusively by apical extension (Sudbery, 2011). Each of these growth forms is associated with changes in cell cycle regulation, expression of morphology-specific gene sets and differences in host immune response. Therefore, morphogenesis of C. albicans impacts directly on pathogenesis (Gow and Hube, 2012). Most monomorphic mutants are attenuated in virulence (Sudbery et al., 2004). C. albicans also forms asexual chlamydospores which are seldom, if ever, observed in human tissues.
Another important cell type in C. albicans is the mating competent opaque cell. The white-opaque phenotypic switch was first described in the 1980s (Slutsky et al., 1985, 1987) and later shown to be critically important in determining mating competence of opposite mating types (Hull and Johnson, 1999; Hull et al., 2000; Magee and Magee, 2000). Several transcription factors including Wor1, Czf1, Wor2, and Efg1 play important roles in controlling white-opaque switching (Miller and Johnson, 2002; Zordan et al., 2006). Diploid strains will mate if partners are in the opaque form
and homozygous (aa or aa) for the MLT (mating-type-like) locus
(Miller and Johnson, 2002), and mating partners form elongated conjugation tubes called shmoos that undergo chemotropism and fusion (Lockhart et al., 2003). After fusion, the tetraploid nucleus undergoes concerted chromosome loss to regenerate the diploid

Of the pathogenic Candida species, C. albicans, C. tropicalis and C. parapsilosis are diploid species belonging to the so-called ‘‘CTG clade’’, characterized by a somatic CTG codon reassignment in their genomes. The CTG codon encodes serine and not leucine and therefore results in the fungus mistranslating heterologous recombinant proteins (Santos and Tuite, 1995). This discovery paved the way for generation of codon-corrected reporter genes and genetic markers required for molecular genetic analyses. C. glabrata and C. krusei are more distant relatives of the CTG clade species. They are haploid and more closely related to S. cerevisiae than to C. albicans. Members of the CTG clade have not undergone an ancient whole genome duplication, which significantly shaped the genomes of the S. cerevisiae group of fungi.
C. albicans is typical of most pathogenic Candida species in having a genome size of around 6000 predicted genes (Table 2) (Butler et al., 2009). The nearest phylogenetic relative to C. albicans is C. dubliniensis, yet this species is considerably less virulent than C. albicans and many more distantly related Candida species (Jackson et al., 2009). Expansion of certain virulence-associated gene families often occurs in the sub-telomeric regions of Candida species – such as with the EPA gene family of adhesins of C. glabrata (Kaur et al., 2007). The genetic diversity of such cell wall proteins within a species is often further increased by expansion of trinucleotide repeat regions within rapidly evolving genes. Also, a number of gene families are clearly enriched in pathogenic species of Candida including those encoding classes of cell wall proteins, iron assimilation properties and major facilitator and other transporters (Butler et al., 2009). Genetic diversity within and between species is further increased by recombination events that have taken place between Major Repeat Sequences (MRS) and previous retrotransposons. Other chromosome changes such as the formation of partially aneuploid strains and strains with isochromosomes have been shown to affect antifungal drug sensitivities (Selmecki et al., 2006).
3.2. The toolbox for C. albicans
C. albicans diploidy and codon usage present significant technical difficulties in performing functional genetics (see above). Consequently, bespoke nutritional markers used in gene disruption protocols and reporter genes had to be developed for use in this organism (Table 3). Genes from other organisms naturally devoid of CTG codons or which have been codon-corrected, or fully codon-optimized genes, have been used to enable heterologous expression (Fonzi and Irwin, 1993; Gerami-Nejad et al., 2009).
The progenitor method of gene disruption methodologies in C. albicans has been based on the so-called ‘‘Ura-blaster’’ method (Fonzi and Irwin, 1993). The use of this system is associated with some well recognized problems, mainly related to the issue that null mutants are heterozygous for URA3 and the Ura3 gene product

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can become limiting in vivo affecting virulence (Brand et al., 2004). In order to circumvent this, URA3 can be integrated into a high expression locus and alternative genetically marked strains have been generated based on complementation of leu2, his1 and arg4 which do not have compromised virulence. Other C. albicans strains have been generated that use non-nutritional-based selectable markers such as nourseothricin and flipper (FLP) or Cre-lox recombinase to rapidly excise selectable markers (Table 3). In addition,

several systems have been developed that use regulated promoters to control expression of a single functional allele (Samaranayake and Hanes, 2011).
A wide range of in vitro, ex vivo and in vivo assays have been developed to assess the virulence of wild type and mutant strains of Candida species (Table 3). In vitro assays include exposure to primary cell cultures or cell lines of epithelial immune cells and the measurement of cell damage by the release of chromium-51 or

Fig. 2. Pathogenicity assays. (A) Laboratory rodents used as models for fungal research of A. fumigatus, C. albicans and F. oxysporum. Immunosuppression of the host can be achieved via cortisone or corticosteroid treatment to mimic a leukopenia. Ways of infection include injection in the tail vein to generate a disseminated infection, inhalation directly from spore suspension and respiration in an aerosol chamber to generate pulmonary infection. (B) C. albicans invading the chicken embryo chorioallantoic membrane (CAM) (adapted from Gow et al., 2003). (C) Larvae of the greater wax moth (Galleria mellonella) used to investigate virulence of A. fumigatus, C. albicans and F. oxysporum. Tipically, the infection is performed via micro-injection in the posterior pseudopod. Progression of the fungal infection is associated with melanization of the larvae. (D) Embryonated eggs used as infection models in A. fumigatus and C. albicans research. The egg must be perforated at the blunt end and on the side, where an artificial air chamber is then generated applying a negative pressure from the blunt end hole. After perforation of the shell membrane, the inoculum is injected into the artificial air chamber onto the chorioallantoic chamber using a sterile syringe. The holes can then be sealed with paraffin. Art ch, artificial air chamber S, shell; Amc, amniotic cavity; Sm, shell membrane; As, air sac; Ys, yolk sac; Chm chorioallantoic membrane; Alc, allantoic cavity; Alb, albumin. (E–G) F. oxysporum assays. (E) Tomato plant root assay. Two week old tomato seedlings (cultivar Money Maker) are inoculated with F. oxysporum strains by immersing the roots in a microconidial suspension for 30 min, planted in vermiculite and incubated in a growth chamber at 28 °C. Evaluation is performed using a disease index for Fusarium vascular wilt going from 1 = healthy plant to 5 = dead plant. (F-G) Invasive growth assay on living fruit tissue. Apple fruits (F) or tomato fruits (G) are inoculated with F. oxysporum strains and incubated in a humid chamber at 28 °C for 3 days. (H–K) M. oryzae assays. (H) Typical oval-shaped lesions on rice leaves, cultivar (cv) Co-39, generated after spray inoculation of the pathogen (strain Guy11) (kindly donated by Dr. Michael J. Kershaw). (I) Invasive hyphae on rice sheath at 29 h post-inoculation obtained from a rice leaf sheath assay. (J) Spray inoculation of barley, 6 days post-inoculation (kindly donated by Dr. Michael J. Kershaw). (K) Drop inoculation assay on barley, 6 days post-inoculation. (L) U. maydis strains are injected into 7-dayold maize seedlings. Disease symptoms are scored according to different categories based on tumor size. 1. Chlorosis. 2. Small swelling at ligula or stem. 3. Small tumors on the leaves. 4. Large tumors on leaves. 5. Heavy tumors on the base of the stem and/or dead plant. (M–Q) Z. tritici. Symptoms on wheat leaves. (M) Natural infection on the third leaf in field tests (cv. CYMMIT). (N) Pycnidia produced by the strain IPO94269 on wheat leaves (cv. Obelisk), 21 days post inoculation (dpi). (O) Close up of (N) showing pycnidia and cirrhi. (P) Inoculation of Z. tritici onto the second leaf using a paintbrush in greenhouse. (Q) Symptoms produced by different isolates of Z. tritici on flag leaves at 35 dpi: isolates INRA08-FS0002 (1) and isolate IPO323 (2) on cv. Apache (E) (Suffert et al., 2013). Images kindly donated by Dr. Frederic Suffert and Dr. Thierry C. Marcel.

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Fungal Genetics and Biology